Information flow within the nervous system requires the perpetuation of ionic gradients along neurons. Myelin is a lipid-rich dielectric substance that ensheathes axons and provides insulation. The nervous system contains high levels of myelin, which is especially enriched where many myelinated axons are bundled together, collectively called “white matter”, as opposed to “grey matter”. Because non-nervous system tissues lack myelin, the presence of myelin can distinguish nerve tissue from other tissue types, such as the spinal cord and spinal nerve roots from non-nervous elements of the vertebral column, white matter from grey matter in the brain, and peripheral nerves from muscle tissue. The ability to qualitatively or quantitatively visualize myelin, either in vivo or in vitro, offers researchers and clinicians important diagnostic and treatment tools. For example, the ability to visually identify peripheral nerves during surgery assists surgeons in avoiding cutting or damaging nerves.
Previous efforts in image-guided surgery of nerves utilized modalities that would not require contrast agents or fluorescent labeling of axons by retrograde transport. Challenges in nerve labeling by a retrograde transport approach include the generation of ambiguous signals, the requirement for nerve-stimulation as well as the additional expense of time and effort. In a retrograde transport labeling approach, the labeling efficiency would depend on the exact area of injection and nerves might not be visualized if they fail to take up the contrast agents.
The availability of myelin markers, myelin labeling dyes and myelin-labeling methodologies is paramount in advancing anatomical studies in neuronal research, including neural stem cell research, development of various therapies, and availability of putative animal models of myelin-associated neuropathies. In vivo myelin imaging of the spinal cord assists clinicians in the diagnosis and treatment of spinal cord pathology, such as nerve compression or herniated discs, as well as in diagnosing myelin-associated neuropathies, such as multiple sclerosis and Alzheimer's disease, which stems from damage to myelin within the nervous system. The ability to measure the degree of myelination in vivo in patients with such conditions would aid diagnosing and prognosing myelin-associated neuropathies. Syndromes such as cervical radiculopathy, sciatica, intervertebral disc herniation, and root compression are caused by compression of nerves primarily from tumors or other lesions, which usually results in back or neck pain. The ability to image and identify the source of chronic neck or back pain could enable surgeons to effectively treat these syndromes.
The existing myelin-labeling methodologies include the use of commercially available FluoroMyelin dyes for identification of high myelin content tissues. However, except for a few dyes such as bis-styrene-arylene dyes such as 1,4-bis(p-aminostyryl)-2-methoxy benzene, and (E,E)-1,4-bis(4′-aminostyryl)-2-dimethoxy-benzene, most of the known dyes are unable to cross the blood nerve bather (BNB) or blood brain barrier (BBB).
Myelin is a protein and lipid-rich matrix formed by oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). Because two different cell types in CNS and PNS produce myelin, namely oligodendrocytes and Schwann cells respectively, there are similarities and differences in protein and lipid composition depending on the source of myelin. In both instances, myelin is composed of about 80% lipid fraction and about 20% protein fraction. The lipid fraction in myelin contains cholesterol, cholesterol ester, cerebroside, sulfatide, sphingomyelin, phosphotidylethanolomine, phosphotidylcholine, phosphotidylserine, phosphotidylinositol, triacylglycerol, and diacylglycerol. The protein fraction is composed of several proteins, which include myelin basic protein (MBP), peripheral myelin protein 22 (PMP22), connexin 32 and myelin-associated glycoprotein (MAG), which are produced by both PNS and CNS cells. The myelin protein zero (MPZ) and proteolipid protein produced by the PNS and CNS cells respectively.
The MBP is a major protein component of myelin at 5%-15%, which translates into about 5 mM concentration of MBP. The interaction of MBP with lipids may cause conformational variability and may be critical for exercising its function. An agent that selectively binds to MBP may result in improvements in myelin staining and thereby aid in nerve visualization. Nerve visualization may further be improved through optimal elimination of unbound and nonspecifically bound dye, and improved optical properties allowing for enhanced contrast between myelin and the surrounding tissue. Optical imaging in the near infrared range (NIR), between 700-900 nm, is ideally suited for visualization of myelin in vivo, as the absorption of water, hemoglobin, and lipid are minimal resulting in reduced scatter and improved photon penetration. However, a dye that excites and emits in the visible region is also advantageous. In particular, a specific, targeted fluorophore with a large Stoke shift can provide a high signal-to-background despite operating in the visible region. Furthermore, this approach is complementary to NIR imaging, and does not interfere with NIR fluorescence if multi-channel molecular imaging is desired.